Preservative compositions and methods for mushrooms

ABSTRACT

Preservative compositions using toxicologically acceptable ingredients, and employing a pH of 9.0 or above for at least part of the process, for controlling the growth of spoilage bacteria and for preventing unwanted color changes in fresh and processed mushrooms. Aqueous solutions of preservatives are prepared and applied in multiple stages to the mushrooms, by spraying or immersion. More specifically, disclosed is a method for preserving fresh and processed mushrooms, comprising the steps of: contacting the mushrooms with an antimicrobial buffer solution having a pH of from about 9.5 to about 11.0; and rinsing the mushrooms one or more times immediately after the contacting step with pH-neutralizing buffer solutions of erythorbic acid and sodium erythorbate, in ratios of about 1:4, with a sufficient pH to return the mushrooms to the mushroom physiological pH of about 6.5.

This application claims the benefits of co-pending U.S. ProvisionalPatent Application Ser. No. 60/060,670 filed Oct. 2, 1997.

FIELD OF THE INVENTION

The present invention relates generally to methods for retardingbacterial spoilage and other unwanted quality changes in fresh andprocessed mushrooms that are intended for ingestion by humans and loweranimals, and more specifically to preservative compositions, especiallythose employing a pH of 9.0 or above as part of the process, which areespecially suitable for practicing said methods.

BACKGROUND OF THE INVENTION

Consumers identify whiteness and cleanliness of fresh white buttonmushrooms (Agaricus bisporus) as the principal factors determining thequality thereof (McConnell, 1991; Beelman, 1987; Schisler, 1983;Barendse, 1984; Wuest, 1981). Consumers prefer to purchase mushroomswhich are bright white and free of casing material, compost, or otherunwanted particulate contaminants clinging to the surfaces thereof(McConnell, 1991).

Commercial mushroom cultivation practices, typically growing mushroomsin straw-bedded horse manure compost covered with a fine layer of peator other “casing material,” yields mushrooms with unwanted particulatecontaminants clinging to the mushroom cap and other surfaces, giving anundesirable appearance (McConnell, 1991). Moreover, mushrooms aretypically harvested by hand, introducing a source of contamination withfluorescent pseudomonads and other spoilage organisms, leading toaccelerated tissue decay and discoloration (McConnell, 1991).

Mushroom discoloration (browning and purple blotch) occurs when apolyphenol oxidase enzyme (tyrosinase), which naturally occurs at highlevels in mushroom cap cuticle (surface) tissue, interacts with phenolicsubstrates, also naturally occurring in mushroom tissue, to produce thebrown pigment melanin. In healthy, intact mushroom tissue, the enzymeand its substrates are located in separate subcellular compartments, andare therefore prevented from reacting to form colored pigments.Unfortunately, mushroom tissue is highly susceptible to damage bybacterial action or by physical handling, and this damage allows thebrowning enzyme and its substrates to interact, resulting in unwantedcolor changes in the mushroom tissue.

It would be highly desirable, therefore, to provide a commercial,toxicologically acceptable preservative treatment to prevent bacterialdamage to mushroom tissue, indirectly preventing discoloration, and toinhibit directly the polyphenol oxidase-mediated browning reaction.Moreover, it would be especially desirable to introduce thesepreservatives to mushrooms in the form of a spray or wash which wouldremove compost, casing material, and other unwanted particulate materialcling to mushroom surfaces.

Prior to 1986, aqueous solutions of sulfite, particularly sodiummetabisulfite, were used to wash mushrooms for the purpose of removingunwanted particulate matter, and to enhance mushroom whiteness. In 1986,however, the U.S. FDA banned the application of sulfite compounds tofresh mushrooms, due to severe allergic reactions to sulfite amongcertain asthmatics.

Following the ban on sulfite compounds for processing of freshmushrooms, there have been several efforts to develop wash solutions foruse as a suitable replacement for sulfites. While sulfite treatmentyields mushrooms of excellent initial whiteness and overall quality, itdoes not inhibit the growth of spoilage bacteria. Therefore, the qualityimprovement brought about by sulfite use is transitory. After 3 days ofrefrigerated storage, bacterial decay of sulfited mushrooms becomesevident. Traditionally, this was not a concern to mushroom growers,because sulfite washes were inexpensive, effective at removing unwantedparticulates, and gave excellent initial quality.

The banning of sulfite washes, however, gave researchers incentive notonly to find a suitable sulfite replacement, but also to improve uponsulfite washes by developing a preservative treatment which would extendwashed mushroom shelf life beyond that attainable by sulfiting, andwhich would improve storage quality over that of sulfited mushrooms.McConnell (1991) developed an aqueous preservative wash solutioncontaining 10,000 parts per million (ppm) hydrogen peroxide and 1000 ppmcalcium disodium EDTA. The hydrogen peroxide serves as an antimicrobialagent, while EDTA enhances antimicrobial activity and directlyinterferes with the enzymatic browning reactions. Copper is a functionalcofactor of the mushroom browning enzyme, tyrosinase, and tyrosinaseactivity is dependent upon copper availability. EDTA binds copper morereadily than does tyrosinase, thereby sequestering copper and reducingtyrosinase activity and associated discoloration of mushroom tissue.

Hydrogen peroxide acts as a bactericide by causing oxidative damage toDNA and other cellular constituents. Sapers (1994) adapted McConnell's(1991) hydrogen peroxide treatment, incorporating hydrogen peroxide intoa two-stage mushroom wash, employing 10,000 ppm hydrogen peroxide in thefirst stage and 2.25% or 4.5% sodium erythorbate, 0.2% cysteine-HCL, and500 ppm or 1000 ppm EDTA in aqueous solution in the second stage.Hydrogen peroxide treatments typically yielded mushrooms nearly as whiteas sulfited mushrooms initially, and whiteness surpassed that ofsulfited mushrooms after 1-2 days of storage at 12° C., and shelf lifewas dramatically improved (McConnell, 1991). Hydrogen peroxide, however,is not currently approved for treatment of fresh produce. More efficacyand safety data are required. Moreover, as the browning reaction itselfis oxidative, it would be advantageous to employ a non-oxidative agent,rather than a strong oxidizer such as hydrogen peroxide, for controllingbacterial growth.

SUMMARY OF THE INVENTION

The present invention provides a sulfite alternative employing high pH(preferably 10.5-11.0) to control bacterial growth on mushrooms, andbrowning inhibitors to minimize enzymatic browning of mushroom tissue.

High pH (9.0 or above) has been shown to be effective for controllingthe growth of bacteria in egg washwater (Catalano and Knabel, 1994). Thepresent invention adapts high-pH solutions as an antimicrobial washtreatment for fresh mushrooms, to prevent bacterial decay of mushroomtissue and resultant tissue browning. With their high susceptibility totissue damage, mushrooms represent a unique application of high-pHpreservative treatments. Solution exposure time must be carefullycontrolled, to optimize bacterial destruction while avoidingcounterproductive overexposure of mushrooms to extremes of pH, resultingin chemical damage to tissue. Thus, the present invention comprises amultiple (two- or three-) stage wash procedure, with an initial high-pHantimicrobial step, followed by one or more pH neutralization/browninginhibitor washes, with an erythorbic acid/sodium erythorbate buffer withEDTA added, for example.

The present invention provides a high-pH treatment for the control ofbacterial spoilage of mushrooms. A first-stage, high-pH wash destroysbacteria, but might also directly damage mushroom tissue. This isavoided, however, if mushroom exposure time to the high-pH solution isbrief and is followed immediately by a second-stage neutralizationbuffer, consisting primarily of the enzymatic browning inhibitorserythorbic acid and sodium erythorbate.

Solutions of varying concentrations of trisodium phosphate (TSP) orsodium bicarbonate were adjusted to pH 11.0 and reacted with equalvolumes of erythorbic acid/sodium erythorbate browning inhibitorsolutions, to screen for combinations yielding a final pH in themushroom physiological range. Solutions with the desired bufferingcapacities were screened for effectiveness in vivo in mushroom washingtrials. Reflectance colorimetry and visual inspection for bacterialblotch and other defects were used to determine mushroom quality. A0.05M sodium bicarbonate buffer wash at pH 10.5-11.0, followed by a 0.6%erythorbic acid/2.4% sodium erythorbate wash yielded initial qualitynearly as high as that obtained by sulfite treatment, and far exceededthe performance of sulfite treatment on days 3, 6, and 9 of storage.

With the pH 11.0/3% erythorbate treatment as a starting point, furtherexperiments were designed to optimize the process, examining the effectsof varying mushroom exposure time to wash solutions, varying solutiontemperatures, and the addition of EDTA and calcium chloride to thesecond-stage wash solution. Optimum mushroom quality and shelf life wereobtained when mushrooms were washed in the high-pH solution for 30s at25° C., and in the erythorbic acid/sodium erythorbate solution for 60sat 10° C. Addition of 1000 ppm calcium-disodium EDTA and 1000 ppmcalcium chloride to the second-stage wash further improved mushroomquality. The high-pH/erythorbate treatment with EDTA and calciumchloride equaled or exceeded the initial quality yielded by sulfitetreatment, and far exceeded the performance of sulfite treatment on days3, 6, and 9 of storage. Thus optimized high-pH treatment also equaled orexceeded the performance of a hydrogen peroxide/EDTA treatment on eachday of evaluation, and was as effective as an antimicrobial.

In addition to improving the quality and shelf life of fresh mushrooms,the high pH/erythorbate wash treatment has applications in canning andin freezing. High-pH treatment prior to canning resulted in better(lighter) color than did sulfite or water washing before canning.Mushrooms treated with high pH prior to freezing were much whiterthroughout frozen storage than mushrooms washed in water or a sodiumsulfite solution.

A principal objective of the present invention is to provide a practicalwash treatment that will yield mushrooms as white as sulfite-treatedmushrooms initially, while also suppressing bacterial growth, extendingshelf life, and improving storage quality.

It is a principal object of the present invention to apply high pHbactericidal solutions to mushrooms followed by neutralization ofmushroom pH and introduction of browning inhibitors, to preventbacterial decay and mushroom tissue discoloration.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a chart illustrating the effect of adding EDTA and calciumchloride to the second-stage wash solution of the high-pH treatment.Within each day treatments with the same letter are not different atp<0.05.

FIG. 2 is a chart illustrating the effect of retention time in washsolution on color of hybrid off-white mushrooms. Within each day,treatments with the same letter are not significantly different at the5% level.

FIG. 3 is a chart illustrating the effect of retention times in washsolutions on the color of hybrid off-white mushrooms. Slopes with thesame letter are not significantly different at the 5% level.

FIG. 4 is a chart illustrating the effect of wash solution temperatureon the quality of hybrid off-white mushrooms. Within each day,treatments with the same letter are not significantly different at the5% level.

FIG. 5 is a chart illustrating the effect of wash solution temperatureson the quality of hybrid off-white mushrooms. Slopes with the sameletter are not significantly different at the 5% level.

FIG. 6 is a chart illustrating the effect of first-stage wash solutionpH on the color of hybrid off-white mushrooms. Within each day,treatments with the same letter are not significantly different at the5% level.

FIG. 7 is a chart illustrating the effect of first-stage wash solutionpH on the color of hybrid off-white mushrooms. Slopes with the sameletters (within parentheses) are not different at p<0.05.

FIG. 8 is a chart illustrating the effect of first-stage wash solutionpH on the color of hybrid off-white mushrooms. Within each day,treatments with the same letter are not significantly different at the5% level.

FIG. 9 is a chart illustrating the effect of first-stage wash solutionbuffering capacity on hybrid off-white mushroom color. Slopes with thesame letter are not significantly different at p<0.05.

FIG. 10 is a chart illustrating the effect of erythorbic acid/sodiumerythorbate concentration on color of hybrid off-white mushrooms. Withineach day, treatments with the same letter are not significantlydifferent at the 5% level.

FIG. 11 is a chart illustrating the effect of erythorbic acid/sodiumerythorbate concentration of hybrid off-white mushrooms. Slopes with thesame letter are not different at the 5% level.

FIG. 12 is a chart illustrating the comparison of aerobic plate count onmushrooms from four different treatments. Within each day of evaluation,treatments with the same letter were not different at the 5% level.

FIG. 13 is a chart illustrating the effect of mushroom holding times inwash solutions and solution temperatures on aerobic plate counts. Withineach day, treatments with the same letter were not different at the 5%level.

FIG. 14 is a chart illustrating the effectiveness of high-pH, sulfite,and water wash treatments at maintaining whiteness. Within each day,treatments with the same letter were not different at the 5% level.

FIG. 15 is a chart illustrating the effectiveness of high-pH, sulfite,and water wash treatments at maintaining whiteness over time. The slopewith the asterisk is different from the others at the 5% level.

FIG. 16 is a chart illustrating the effectiveness of three treatments atmaximizing whiteness of canned mushrooms, after one week of storage.Treatments with the same letter are not different at the 5% level.

FIG. 17 is a chart illustrating the canning yield of three treatments,expressed on a fresh weight basis. Treatments with the same letter arenot different at the 5% level.

FIG. 18 is a chart illustrating the effectiveness of three washtreatments at maintaining whiteness of mushrooms stored at −10 C. Withineach week, treatments with the same letter were not different at the 5%level.

FIG. 19 is a chart illustrating the effectiveness of three washtreatments at maintaining whiteness of mushrooms stored at −10 C. Withineach week, treatments with the same letter were not different at the 5%level.

FIG. 20 is a chart illustrating the change in mushroom color with re-useof wash solutions. The sulfite treatment showed a decline in color,while the high-pH treatment did not, at the 5% level.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Harvesting of theMushrooms

Hybrid off-white (U1) mushrooms were grown at the Mushroom TestDemonstration Facility (MTDF) of the Pennsylvania State University, ontraditional horse manure-based compost, using standard MTDF practice.Mushrooms were harvested early in the morning on the day of eachexperiment. Twice as many mushrooms as were needed for washing wereobtained from those picked. Mushrooms were selected for washing based onsize, freedom from major blemishes (bruising, gouges), disease (blotchor verticillium), and for maturity (unstretched veils). Only first andsecond flush mushrooms were used; and, within a given experiment,mushrooms were obtained from a single flush and growing room. Mushroomswere stored at 4° C., randomly assigned to treatment lots, and washedwithin 8 hours of picking. Almost all of the wash treatments testedconsisted of two stages: a first-stage, high-pH antimicrobial wash(typically, a pH 10.0-11.0 sodium bicarbonate buffer), followed by asecond-stage neutralization and preservative wash (typically, a mixtureof erythorbic acid, sodium erythorbate, calcium chloride, and EDTA).Since high pH was employed as the principal antimicrobial factor, it wasnecessary to neutralize pH in the second wash stage, to minimizemushroom tissue damage and resultant acceleration of enzymatic browning.

Initially, two solutions were prepared at pH 11.0, the minimum suggestedpH for useful antimicrobial action: a 0.05M sodium bicarbonate solution(pH 8.25) adjusted to pH 11.0 with 1.0N sodium hydroxide, and a 1%tribasic sodium phosphate solution (pH 11.74) adjusted to pH 11.0 with42.5% phosphoric acid.

Second stage, neutralization solutions were prepared from stocksolutions of 1%, 2% 3%, and 4%, each, of erythorbic acid and sodiumerythorbate. The pH of these stock solutions was measured singly and invarying erythorbic acid:sodium erythorbate ratios, to give severaldifferent formulations at each total solute concentration (1%, 2%, 3%,and 4%).

Neutralization solutions were then combined with equal volumes of pH11.0 solutions, and the final pH of each mixture was recorded. Resultswere screened for combinations yielding final pH in the range of6.50-8.00, i.e., close to mushroom physiological pH, approximately 6.5.Neutralization solutions tested are given in Table 1, with pHmeasurements alone and in mixture with equal volumes of pH 11.0solutions. All pH measurements were made using a Beckman φ 40 pH meter(Beckman Instruments, Inc., Fullerton, Calif.) standardized with FisherCertified ACS pH 4, 7, and 10 buffers (Fisher Scientific, Inc., FairLawn, N.J.). Solutions yielding final pH within the target range werethen used in mushroom washing trials, to determine effectiveness atmaximizing shelf life and optimizing mushroom color (whiteness).

Washing Procedure

Treatment solutions were prepared with deionized (reverse-osmosis) waterand allowed to equilibrate to the desired temperature immediately beforewashing. Typically, the first, high-pH stage of a two-stage washtreatment was adjusted to 25° C., while the second, neutralization stagewas chilled to 10° C. Chemical compounds used in wash solutions arelisted in Table 2. Except in experiments where wash duration was anexperimental variable, total washing time was 90 seconds: 30 seconds forstage one, and 60 seconds for stage two of two-stage, high-pHtreatments, and 90 seconds for single-stage sulfite and deionized(reverse-osmosis) water control lots.

Mushrooms were washed in 3.5-liter polyethylene buckets, at the ratio of300 g mushrooms per liter of wash solution, agitated gently by hand,using a stainless steel slotted mixing spoon, at the rate of 30 timesper minute, and drained in polyethylene colanders. Control mushrooms,treated with a single-stage wash, were transferred to colanders after 30seconds and immediately re-immersed in the wash solution, to simulatethe handling of mushrooms is two-stage treatments.

Washed mushrooms were drained for 5 minutes at room temperature, andcolanders were placed in ⅙-size brown paper grocery bags, to preventexcessive moisture loss during overnight holding, making sure that bagsdid not come into contact with mushrooms. Bags were folded over 10-12 cmfrom the top, to close, and bagged mushrooms were placed in a 4° C.cooler and held overnight before overwrap packaging and initial colordetermination.

Packaging

After overnight storage at 4° C., mushrooms were removed from bags, andeach treatment lot was randomly divided into four sublots of six capseach, labeled “Day 0,” “Day 3,”“Day 6,” and “Day 9.” Mushrooms were thenpackaged by sublot, caps up, in linear-polystyrene tills. “Day 0”mushrooms were evaluated immediately, and the remaining tills wereoverwrapped with 60-gauge, PWMF Vitafilm polyvinylchloride film (TheGoodyear Tire and Rubber Co., Akron, Ohio), for shelf-life evaluationafter 3, 6, and 9 days of storage. A mild heat-sealing treatment wasapplied to the overwrap. Two 3-mm holes were made through the overwrap,at opposite corners of each package, using self-adhesive labels appliedto the overwrap to keep the holes open, to ensure that an aerobicenvironment was maintained during storage.

Day 3, 6, and 9 sublots were stored in a 12° C., environmental chamber(Lunaire Environmental, Inc., Williamsport, Pa.), with four packages pertreatment for sampling on each day of shelf-life evaluation.

Color Measurements

Wash treatment effectiveness at maintaining whiteness and retardingpost-harvest browning was determined by measuring mushroom cap color ondays 0, 3, 6, and 9 of storage. Color was measured at three locations onthe surface of each mushroom cap, using a tristimulus calorimeter(Chromameter Model CR-200, Minolta Corp., Ramsey, N.J.). The Chromameterwas calibrated using the standard white calibration plate supplied withthe instrument, and L*a*b color coordinates were used for allmeasurements. A target color of L=97.00, a=−2.00, and b=0.00 was used asa reference standard for internal calculation of overall color deviation(Delta E) from that of the “ideal white mushroom” (Solomon, 1991).

Experiments were structured in a randomized complete block design. Meanwhiteness (L-value) and overall color change (Delta E) values wereinternally calculated for each of the four replicates of each treatmenton each day of evaluation, to give a total of four data points pertreatment per day. L and Delta E values were analyzed using one-wayANOVA, and means were separated via Fisher's ProtectedLeast-Significant-Difference, with StatView 512+ software (BrainPower,Inc., Calabasas, Calif.).

Bacterial Analysis

Wash treatments yielding the best color (highest L-value, lowest DeltaE), initially and over a 9-day shelf life, were screened to determineeffectiveness at controlling bacterial growth on the mushroom capsurface. Mushrooms were prepared and washed as in the shelf-life colorexperiments, and an additional 400 g of mushrooms were randomly sampledfrom each replicate of each treatment, for each day of analysis(0,3,6,9).

Each 400 g sample was randomly divided into two lots of approximately200 g, one for total aerobic plate count (APC) on Eugon agar (DifcoLaboratories, Detroit, Mich.), and the other for coliform count onviolet red bile agar (VRBA) (Difco Laboratories, Detroit, Mich.). Eachlot (approximately 200 g) was homogenized with 200 ml of 0.1% peptone ina sterile Waring blender for 1 minute, modifying the procedure of Simons(1994). Mushroom homogenate was serially diluted using 11 ml transfers,followed by 0.1 ml transfers onto duplicate spread plates containingEugon agar or VRBA. The plates were incubated at 32° C. for 48 hours.

Texture (Firmness) Measurements

Texture was measured the day after washing, using a TA XT2 TextureAnalyzer (Stable Micro Systems, Surrey, England) fitted with a conicalprobe. Penetration depth was set at 0.4 mm. Three readings were takenper mushroom cap, and results were displayed using Stable Micro Systems'XTRA software package.

Canning and Freezing

Washed mushrooms were prepared as canned and frozen products, toevaluate wash treatment effects on canned mushroom color and yield, andon frozen mushroom color. A 60 lb. (27.25 kg) sample of hybrid off-white(U-1) mushrooms was obtained from normal crops grown at the MushroomTest Demonstration Facility (MTDF), the same morning on the day ofwashing. Mushrooms were selected from the 27.25 kg sample on the basisof size, maturity (unstretched veils) and freedom from disease, bruisingand other major blemishes, and randomly assigned to three treatment lotsof 4.5 kg each.

One treatment lot served as a water-washed control, in which mushroomswere washed in 20° C. deionized (reverse-osmosis) water for 90 seconds,at the ratio of 300 g mushrooms per liter of wash solution. Mushroomswere gently agitated by hand, with a stainless steel slotted spoon, 30times per minute. The second treatment lot was washed in a 20° C.solution of 1000 ppm sodium meta-bisulfite for 90 seconds, at the ratioof 300 g mushrooms per liter of solution, and agitated as in the watercontrol. Water and sulfite control mushrooms were transferred to apolyethylene colander after 30 seconds and then immediately placed backinto the wash solution, to simulate the handling of mushrooms in thetwo-stage wash experimental treatment lots.

Experimental treatment mushrooms were washed for 30 seconds in a 0.05Msodium bicarbonate solution, preadjusted to pH 11.0 with 1.0N sodiumhydroxide, at 25° C. After 30 seconds, mushrooms were immediatelytransferred to a 10° C. neutralization wash solution of 6 g/l erythorbicacid, 24 g/l sodium erythorbate, and 1000 ppm calcium-disodium EDTA, at10° C., and immersed for an additional 60 seconds, for a total wash timeof 90 seconds. In both wash stages, mushrooms were washed at the ratioof 300 g per liter of solution, and agitated by hand with a slottedstainless steel spoon, 30 times per minute, as in water and sulfitecontrol treatments.

All mushrooms were drained in polyethylene colanders for 5 minutes atroom temperature, with five colanders of 900 g each, on a fresh weightbasis, for each of the three treatments. One colander from eachtreatment was randomly selected for immediate freezing. Mushrooms to befrozen were randomly separated into six lots of 150 g each, sealed inquart-size polyethylene freezer bags, and immediately placed in thewalk-in freezer at ˜18° C. Color readings and bacterial counts weredetermined at 2, 4, 6, 8, 10, and 12 weeks of frozen storage, using theprocedures for fresh mushroom evaluation, except that color readingswere collected both while the mushrooms were frozen and after thawing.

The remaining four replicate colanders of 900 g mushrooms from eachtreatment, were placed in ⅙-size grocery bags, as for fresh mushrooms,and stored for 24 h at 12° C., in preparation for canning, simulatingcommercial practice. Each mushroom lot was blanched for 5 minutes inboiling water, using steam-jacketed stainless steel kettles, andpre-blanching and post-blanching weights were recorded.

After blanching, the mushrooms were drained for 2 minutes in a stainlesssteel colander, and drained weights were recorded. For each lot, drainedmushrooms were placed into #211×212 cans. A 40-grain sodium chloridetablet was added to each can; cans were filled to the top with boilingtap water, and cans were closed using a Model 424-IES-00 Closing Machine(American Can Co., Greenwich, Conn.). Canned mushrooms were stored for 7days at room temperature, cans were opened, and color (L-value and DeltaE) and canning yield were determined. Canning yield was calculated bydraining each series of six cans for two minutes in a stainless steelcolander, recording the final drained weight, and calculating percentyield on a fresh weight basis. A single color reading was taken for eachmushroom, for 50 randomly-selected mushrooms per series of six cans.Color (L-value and Delta E) was internally averaged for each series ofcans, for a total of four data points and 200 color readings pertreatment.

Tribasic Sodium Phosphate Trials

In preliminary experiments, solutions of tribasic sodium phosphate(trisodium phosphate, TSP), were used to generate a washwater pH of 11.0or higher, as a one-stage wash or in combination with water or theenzymatic browning inhibitors erythorbic acid or sodium erythorbate, ina second-stage wash solution.

Use of 10% TSP by itself, in a wash lasting 120 seconds, was destructiveto mushroom pileal tissue, yielding a Day 0 whiteness (L) value of 6042, vs. 93.36 for a reverse-osmosis water wash and 95.10 for a 1000 ppmsodium metabisulfite wash (Appendix Table 1). TSP-washed mushrooms weredark brown in color and slimy in texture, compared to the bright white,dry, firm sulfite control mushrooms. Reduction of mushroom exposure timeto TSP from 120 seconds to 60 seconds, followed by areverse-osmosis-water wash of 60 seconds dramatically improved color,giving a day-0, L-value of 80.22.

Replacing water with a 2.25% sodium erythorbate solution in thesecond-stage wash yielded a further improvement in color, to an initial(Day 0) L-value of 89.23. When 2.25% sodium erythorbate was replacedwith an equal concentration of erythorbic acid, initial whiteness washigher still, with a day-0, L-value of 90.71. Increasing erythorbic acidconcentration from 2.25% to 4.50% gave very little improvement in colorthrough day 3, but on day 6, the increased erythorbic acid treatment wasnoticeably better, with an L-value of 89.50, versus 84.12 for the 2.25%erythorbic acid treatment. Reduction of TSP concentration from 10% to 5%in the treatments with water as the second-stage wash improved color ondays 0, 3, and 6.

None of the experimental treatments matched the whiteness of the sulfiteand water controls through Day 3, but the two-stage treatment with 4.50%erythorbic acid as the second-stage wash was significantly better thanthe water-washed control and not significantly different from thesulfite-washed control on Day 6.

Development of a Two-Stage, High-pH/Neutralization Wash Treatment

Results of the trisodium phosphate wash trials indicated that thequality of mushrooms washed in basic-pH antibacterial solutions could beimproved by subsequent transfer to a neutralization solution oferythorbic acid and sodium erythorbate. Erythorbate solutions acted asboth an antioxidant, slowing the enzymatic browning reaction, and anacidulant, returning final mushroom pH to physiological range(approximately 6.5), thus minimizing tissue damage due to exposure tohigh pH.

Solutions of 1%, 2%, 3%, and 4% total erythorbate were prepared, each at4:1, 3:1, 1:1, and 1:3 erythorbic acid; sodium erythorbate ratios.Single 1%, 2%, 3%, and 4% erythorbic acid and sodium erythorbatesolutions were also prepared, for a total 24 test solutions. Solution pHwas measured initially and after mixing with an equal volume of 1%trisodium phosphate at pH 11.0, or with 0.05M sodium bicarbonate at pH11.0. Results are given in Table 1. The buffering capacity of the TSPsolution was greater than that of the sodium bicarbonate solution.Several 2%, 3%, and 4% erythorbic acid/sodium erythorbate combinationseffectively acidified the sodium bicarbonate buffer to physiological pH.Only the most acidic (3:1 erythorbic acid: sodium erythorbate) 4%solution, and single 3% and 4% erythorbic acid solutions acidified theTSP solution to near physiological pH.

Wash solution combinations yielding a final pH within or near themushroom physiological range were screened in wash trials, to determineeffectiveness at maintaining whiteness throughout a 9-day shelf life.Wash solutions were maintained at room temperature (20° C.). Mushroomswere immersed in the pH 11.0; buffer for 120s, followed by immersion inthe erythorbic acid/sodium erythorbate buffer for 60s. The TSP-washedmushrooms were not as white initially and did not maintain whitenessduring storage as well as those washed in sodium bicarbonate (AppendixTable 3).

Mushrooms washed in the pH 11.0, 0.05M sodium bicarbonate buffer,followed by the 0.8% erythorbic acid/3.2% sodium erythorbate buffer,were nearly as white initially (L=90.08) as those washed in the 10,000ppm hydrogen peroxide/1000 ppm calcium-disodium EDTA treatment developedby McConnell (1991), (L=90.48). They were not as white initially asmushrooms washed in a 1000 ppm sodium metabisulfite solution (L=91.56).On day 3, however, the pH 11.0/erythorbate-washed mushrooms were whiter(L=91.78) than either the sulfite-treated mushrooms (L=91.00) or theperoxide-dipped mushrooms (L=90.89). The pH 11.0/erythorbate mushroomscontinued to be the whitest on day 6 and day 9, with the L-valuedifference between treatments increasing with time. The two-stage, pH11.0, 0.05M sodium bicarbonate/0.8% erythorbate+3.2% sodium erythorbatetreatment was used as the reference standard for formula- andprocess-optimization experiments, with the goals of enhancing initialwhiteness to equal or exceed that obtained by sulfite treatment,improving whiteness throughout shelf life, and minimizing ingredientusage.

Addition of EDTA and CaCl₂ to the Second-Stage Wash

McConnell (1991) found that the addition of 1000 ppm calcium-disodiumEDTA enhanced the performance of an antimicrobial. 10,000 ppm hydrogenperoxide wash solution, supporting the findings of Eagon (1984) andShibasaki (1978), that EDTA enhances the effectiveness of antimicrobialagents. In addition, EDTA may inhibit enzymatic browning in mushrooms bysequestering copper, a tyrosinase cofactor (McCord and Kilara, 1983).

The shelf life and quality benefits of adding calcium chloride tomushroom irrigation water have been extensively documented (Kukura,1997, Miklus and Beelman, 1996, Simons, 1994, Solomon et al., 1991,Barden et al., 1990). Guthrie (1984) found that the addition of calciumchloride (10 mM) to Oxine antibacterial solutions enhanced theantibacterial effect and yielded firmer mushrooms.

When 1000 ppm calcium-disodium EDTA and then 1000 ppm calcium chloridewere added to the erythorbic acid/sodium erythorbate stage of the pH11.0/erythorbate wash treatment, there were significant improvements inmushroom whiteness, at p<0.05. The improvement in whiteness was alsonoticeable upon visual inspection. Results are given in FIG. 1 and inTable 3. In the experiment summarized in FIG. 1, mushrooms were held inthe pH-11.0 solution for 60 seconds, followed by 120 seconds in a 4%erythorbate solution. Table 3 represents a separate experiment, in whichthe pH-11.0 wash was 30 seconds, followed by a 60-second wash in a 3%erythorbate solution. The color improvement due to calcium chloride wasgreater for the longer wash time, 120 seconds (FIG. 1), in the 4%erythorbate solution, vs. 60 seconds (Table 3) in the 3% erythorbatesolution. It was subsequently shown, however, that the best overallperformance was yielded by the 30-second pH-11.0 wash, followed by the60-second, 3% erythorbate+1000 ppm EDTA+1000 ppm calcium chloride wash.

Kukura (1997) showed that mushrooms irrigated with tap water pluscalcium chloride were more resistant to discoloration in general, andespecially discoloration due to bruising, than were mushrooms irrigatedwith tap water alone. For mushrooms subjected to bruising treatments,calcium-chloride irrigation was shown to strengthen cell and vacuolemembranes, preventing the leakage of polyphenoloxidase (PPO) substratesfrom the vacuole to the cytoplasm and surrounding medium. Containment ofPPO substrates in the vacuole prevents them from interacting with theenzyme, thus preventing enzymatic browning. Electron microscopy did notreveal the same structural difference between calcium-added andno-calcium treatments when calcium chloride was incorporated into thewash treatment. Mushrooms in this study, however, were not subjected tobruising, and this may explain why the protective effect of calcium wasnot evident in the micrographs of washed-mushroom tissue. There was,however, an improvement in mushrooms whiteness as a result of theaddition of 1000 ppm calcium chloride to the second-stage wash solution(FIG. 1, Table 3).

Calcium chloride addition to the second-stage wash also affectedbacterial populations. On day 0, plate counts were higher forcalcium-treated mushrooms, vs. high-pH, no-calcium mushrooms, at p<0.05(Table 4). By day 9, however, plate counts for high-pH, no-calciummushrooms were significantly higher than counts for high-pH-plus-calciummushrooms. There was no significant difference in plate count betweenthe two high-pH treatments on day 3 and day 6.

Barden et al. (1990) found that bacterial counts were consistently lowerfor mushrooms with 0.5% calcium chloride added to the irrigation waterthan for mushrooms with no calcium chloride added to the irrigationwater. The day 9 plate count results suggest that a similar relationshipbetween calcium and bacterial growth exists at the end of the shelf lifefor mushrooms washed in high-pH solutions containing 0.1% calciumchloride.

Solomon (1989) proposed that improvements in mushroom quality due toCaCl₂ irrigation treatments were the result of surface accumulation ofcalcium, which reduced water activity and bacterial growth, andconcomitantly increased surface light reflectance. This is supported bythe data in Table 3, showing an increase in whiteness between day 0 andday 3, possibly the result of post-washing moisture loss. In thewater-washed control mushrooms, the effect is likely negated by thegreater increase is bacterial numbers between day 0 and day 3 (Table 4).

The higher day 0 bacterial populations for the calcium chloride high-pHwash, vs. the no-added-calcium high-pH wash suggest that, at leastinitially, for high-pH-treated mushrooms, there are effects of calciumon bacterial growth unrelated to the reduction in water activity at thecap surface. Mendonca et al., 1994, concluded that the destruction offood-borne pathogens by high pH involves disruption of the cytoplasmicmembrane. As Ferguson (1984) and Miklus and Beelman (1996) havesuggested that calcium stabilizes biological membranes, it is possiblethat the 0.1% CaCl₂ added to the high-pH wash protected both bacterialcell membranes and mushroom tissue membranes from damage due to high pH.In terms of bacterial survival and growth, however, this appears to beonly an initial effect. After day 0, bacterial counts for calcium-washedmushrooms were found to be lower than or not significantly differentfrom counts for mushrooms washed without calcium. It is possible that,later in storage, the effect of calcium in lowering surface wateractivity predominates.

Time and Temperature Effects Color

Mushroom retention time in the wash solutions and temperatures of thewash solutions were examined, in order to maximize mushroom quality.Changing the holding time in the pH 11.0 buffer from 120s to 60s and inthe erythorbate solution from 60s to 120s, reversing the holding timesfor the two wash solutions, resulted in increased whiteness on days 6and 9 of shelf life. In addition, the rate of discoloration wasdecreased for the mushrooms held for the shorter interval in the high-pHbuffer and for the longer interval in the erythorbate solution. Halvingthe retention times to 30s in the high-pH buffer and 60s in theerythorbate buffer resulted in a further increase in whiteness (FIG. 2),but the rate of discoloration over time (slope of the L-value vs.storage time plot) was not changed from that of the 60s/120s treatment(FIG. 3) The rate of discoloration increased, however, when mushroomswere exposed to the high-pH solution for 120 seconds and only immersedin the neutralization wash for 60 seconds (T-10, FIG. 3).

Temperature data are given in FIGS. 4 and 5. Optimum wash solutiontemperatures were 25° C. for the pH 11.0 buffer and 10° C. for theerythorbate buffer. Increasing the temperature of the high-pH buffer to35° C. decreased whiteness after day 3 of storage, and increased therate of discoloration. Decreasing the temperature of the high-pH bufferto 10° C. had a similar effect on mushroom color. Increasing thetemperature of both solutions, with the high-pH buffer at 35° C. and theerythorbate buffer at 25° C., resulted in a still greater deteriorationin color. All high-pH/erythorbate treatments, however, gave betterquality than washing in either reverse-osmosis water at 10° C. or 1000ppm sodium metabisulfite at 10° C. All mushrooms were equilibrated to 4°C. in a walk-in cooler prior to washing.

Water Uptake

Time and temperature parameters affected mushroom water uptake duringwashing (Table 5). Minimizing water uptake during washing is importantto prevent mushrooms from having a waterlogged appearance. As expected,shorter wash times generally resulted in less water uptake, vs. longerwash times at the same solution temperatures. The relationship betweentemperature of the wash solutions and water uptake was less predictable.Increasing the temperature of the high-pH wash solution from 10° C. to25° C. decreased water uptake (Table 5, Treatment 3 vs. Treatment 7).Further increasing the temperature to 35° C., however, resulted in anincrease, rather than a further decrease, in water uptake (Table 5,Treatment 7 vs. Treatment 5).

Increasing the temperature of the neutralization wash from 10° C. to 25°C. also increased water uptake (Table 5, Treatment 5 vs. Treatment 2).Overall, the time-temperature combination yielding the lowest wateruptake was a 25° C., 30 second high-pH wash followed by a 10° C., 60second neutralization wash.

Texture

Mushroom texture was evaluated, to determine the effects of water uptakeand high pH upon the firmness of mushrooms. There was no significantdifference in firmness between unwashed mushrooms, mushrooms washed inwater or in sodium sulfite, and mushrooms treated with hydrogenperoxide/EDTA or with high-pH/neutralization washes (Table 6).

First-Stage Wash Solution pH vs. Mushroom Quality

The first-stage wash solution was designed to prevent the growth ofbacteria, particularly pseudomonads, on the mushroom cap surface.First-stage wash solution buffers were prepared at pH values of 11.0,10.5, 10.0, 9.5, and 9.0, to determine the optimum pH, with overallmushroom quality the deciding criterion. All treatments used the 30sretention time in the high-pH buffer at 25° C., and the 60s retentiontime in the erythorbate buffer at 10° C., shows to yield the highestquality and the least water uptake. A 0.6% erythorbic acid+2.4% sodiumerythorbate+1000 ppm EDTA+1000 ppm calcium chloride formula was used forall treatments. Results are given in FIGS. 6 and 7.

Mushroom quality generally decreased with decreasing first-stagesolution pH. The pH 10.5 and 11.0 formulations performed best. The pH10.5 and 11.0 formulations were the best performers overall, yieldingmushrooms as white as or whiter than those from other treatments on eachday of evaluation, and having a slower rate of discoloration over time.

The pH 9.5 and 10.0 performed as well as the pH 10.5 and 11.0formulations initially (on day 0). On day 3 and day 6, however, theyyielded mushrooms that were less white than those from the higher-pHtreatments. The pH 9.0-treated mushrooms were not as white initially asthe other high-pH treated mushrooms, and they discolored at a more rapidrate than all but the reverse-osmosis water and sulfite controlmushrooms.

Sulfite-treated mushrooms were as white initially as those from the pH11.0, 10.0, and 9.5 treatments. They discolored at a much higher rate,however, and by day 3, they were not as white as the pH 11.0, 10.0, and9.5-treated mushrooms. By day 6, the pH 9.0-treated mushrooms werewhiter than sulfite-treated mushrooms. Sulfite-treated and water-washedmushrooms discolored at the same rate, but the sulfite-treated mushroomswere whiter initially, and thus on each day of evaluation.

Wash Solution Buffering Capacities vs. Mushroom Quality

The poorer performance of TSP-based treatments, vs. sodiumbicarbonate-based treatments, was attributed to insufficientneutralization (reacidification) of the mushrooms by the erythorbatesolution, due to the greater buffering capacity of the TSP solutions.Conversely, it was possible that the pH 10.0, 9.5, and 9.0-treatedmushrooms were overacidified in the 3.0% erythorbate buffer. To examinethe effects of wash solution buffering capacity on mushroom quality,mushrooms were washed in first-stage high-pH buffers of varying sodiumbicarbonate concentration, and in second-stage buffers of varyingerythorbic acid/sodium erythorbate concentration.

Sodium Bicarbonate Concentration

In the first experiment, the second-stage buffer remained constant, 0.6%erythorbic acid+2.4% sodium erythorbate+1000 ppm EDTA, while first-stagebuffers of varying sodium bicarbonate concentration (0.05, 0.10, 0.25,and 0.50M) were prepared. In all treatments, the first-stage buffer wasadjusted to pH 10.0. A pH of 10.0 was chosen, to determine whether a pH10.0 buffer of increased buffering capacity would maintain whiteness aseffectively as a pH 11.0 buffer of lower buffering capacity (included asa reference treatment). Results are given in FIGS. 8 and 9.

Initial whiteness was the same for all treatments except the watercontrol, which was less white than the rest. On day 3, the pH 10.0treatments with higher sodium bicarbonate concentrations (0.01, 0.25,and 0.50M) were as white as the pH 11.0, 0.05M treatment. The 0.05M, pH10.0 treatment was not as white as the 0.05M, pH 11.0 treatment. On day6, there were no differences in whiteness between any of the pH 10.0treatments and the pH 11.0 treatment. All of the high-pH treatments werewhiter than the sulfite and water controls.

Increasing the buffering capacity of a lower-pH, first-stage washsolution was shown to improve mushroom quality, but the effect was onlyseen in the middle of the storage period. On the first day of storageafter washing and six days after washing, there were no differences inwhiteness between the pH 11.0 treatment and any of the pH 10.0treatments of varying sodium bicarbonate concentration.

Erythorbic Acid/Na Erythorbate Concentration

In this experiment, the first-stage buffer, 0.05M sodium bicarbonate atpH 11.0, was tested in combinations with three different second-stagebuffers:

-   -   1. 0.8% erythorbic acid+3.2% sodium erythorbate+1000 ppm EDTA        (4% total erythorbate).    -   2. 0.6% erythorbic acid+2.4% sodium erythorbate+1000 ppm EDTA        (3% total erythorbate).    -   3. 0.4% erythorbic acid+1.6% sodium erythorbate+1000 ppm EDTA        (2% total erythorbate).

Results are given in FIGS. 10 and 11.

There was no difference in whiteness between mushrooms washed in thethree erythorbate solutions, on any of the days (0,3,6,9) of evaluation.Sulfite control mushrooms were as white as the experimentally treatedmushrooms initially (day 0), but were less white on days 3 and 6. On day9, the 3% and 4% erythorbate-treated mushrooms were still whiter thanthe sulfite-treated mushrooms. Mushrooms treated with 2% erythorbatewere not whiter, at p<0.05, than sulfite-treated mushrooms, on day 9.

Hydrogen peroxide/EDTA-washed mushrooms not as white initially asmushrooms washed in sulfite or in the pH 11.0/3% erythorbate treatment.They were, however, as white as those washed in water, pH 11.0/2%erythorbate, or pH 11.0/4% erythorbate. On days 3, 6, and 9, thehydrogen peroxide/EDTA treatment performed as well as the 2%, 3%, and 4%erythorbate treatments. The rate of discoloration (slope of the L-valuevs. storage-time plot) was not different, at p<0.05, from that of thehigh-pH/erythorbate-treated mushrooms. Sulfite-treated mushroomsdiscolored at a faster rate than all of the other treatments.

In summary, the high-pH treatment with the 3% erythorbate second-stagewash performed best, yielding mushrooms as white as or whiter than thosefrom all other treatments on all four days of evaluation.

Effect of High-pH Treatment on Bacterial Growth

It has been shown in testing to date that, in general, the performanceof a two-stage, high-pH buffer/erythorbate buffer preservative washtreatment increased as the pH of the first-stage buffer increased, asmeasured by mushroom whiteness. In addition to the inhibition ofenzymatic browning by erythorbic acid, sodium erythorbate, and EDTA inthe second-stage buffer, there is an improvement in mushroom shelf lifeand quality as a result of exposure to high pH in the first stage ofwashing. It was hypothesized that this positive effect of high pH onmushroom quality may be due to destruction of spoilage bacteria on themushroom cap surface.

To assess the antimicrobial effect of the high-pH treatment of freshmushrooms, aerobic plate counts were determined for four treatments:

-   -   1. Reverse-osmosis water, 20° C., 90s    -   2. 1000 ppm sodium metabisulfite, 20° C., 90s    -   3. 10 000 ppm hydrogen peroxide+1000 ppm EDTA, 20° C., 90s    -   4. 0.05M sodium bicarbonate at pH 11.0, 25° C., 30s/0.6%        erythorbic acid +2.4% sodium erythorbate+1000 ppm EDTA, 10° C.,        60s. Results are given in FIG. 12. Note that the statistical        groupings differentiate between treatments within a single day        of evaluation, and do not indicate differences in bacterial        populations over time for a single treatment.

Initially and on all three subsequent days of evaluation, the high-pHand the hydrogen peroxide treatments yielded lower bacterial populationsthan did the sulfite and the water control treatments. For all fourtreatments, bacterial populations increased steadily over time. On day0, populations were 2.20×10⁶ CFU/g for the high-pH treatment, 2.34×10⁶CFU/g for the hydrogen peroxide treatment, 5.00×10⁶ CFU/g for the watercontrol, and 5.33×10⁶ CFU/g for the sulfite treatment. On day 6,bacterial numbers for the water and sulfite controls reached 7.20×10⁸and 9.78×10⁸ CFU/g, respectively, while the high-pH and hydrogenperoxide treatments had populations of 1.57×10⁸ and 2.34×10⁸ CFU/g.

The high-pH treatment was as effective as hydrogen-peroxide washing atcontrolling bacterial growth on washed mushrooms. Both yielded lowerbacterial populations than did sulfite treatment or water washing.

Time and Temperature Effects

Wash solution temperatures and mushroom retention times in washsolutions were shown to affect mushroom quality throughout shelf life.These parameters were also investigated microbiologically, to determinetheir effects on mushroom bacterial populations. The same high-pHtreatments were evaluated as for the overall quality experiment:

-   -   1. Reverse-osmosis water, 20° C., 90s    -   2. pH 11.0, 25° C., 30s/3% erythorbate, 10° C., 60s    -   3. pH 11.0, 10° C., 30s/3% erythorbate, 10° C., 60s    -   4. pH 11.0, 25° C., 60s/3% erythorbate, 10° C., 120s    -   5. pH 11.0, 10° C., 60s/3% erythorbate, 10° C., 120s. Aerobic        plate counts were recorded on days 0, 3, and 6. Results are        given in FIG. 13.

On all three days, bacterial populations were lower for the high-pHtreatments, vs. the water control. On day 0, the 25° C./10° C. treatmentwith the 90s total retention time yielded lower bacterial populationsthan did the high-pH treatments with the other time/temperaturecombinations. This treatment also yielded the best shelf-life quality.

On day 3, the 25° C./10° C. treatments at both retention times yieldedlower bacterial populations than did the other treatments. On Day 6, the25° C./10° C., 90s treatment still resulted in lower bacterialpopulations than did all of the other treatments. The longer-retentiontime treatments, at both temperature combinations, yielded thenext-lowest bacterial populations, while the 10° C./10° C., 90streatment gave the highest bacterial population of he high-pHtreatments.

These results, with a greater bacteria kill occurring at 25° C. than at10° C., confirm the findings of Raynor (1997). Teo et al. (1995), andCatalano and Knabel (1994), that the antibacterial effectiveness of ahigh-pH solution is temperature-dependent. Exposure time was also aninfluencing factor, and there was a time-temperature interaction. On day0 and day 6, the 25° C. treatment at 90s total wash time yielded lowerbacterial numbers than did the same treatment at 180s total wash time.This may have been due to a decrease in water uptake and a resultantincreased rate of drying, leaving less surface water available tosupport bacterial growth. At the lower temperature, where bacterialdestruction occurred more slowly, the longer wash time (60s in the pH11.0 wash) resulted in lower bacterial numbers, on day 6, than did theshorter wash time (30s in the pH 11.0 wash). (FIG. 13).

Performance of Optimal High-pH Treatment vs. Sulfite and HydrogenPeroxide Treatments

Sulfite treatment, though banned commercially from use on freshmushrooms, was still the benchmark, in testing to date, for initialmushroom whiteness. Sulfite treatment produced bright, extremely whitemushrooms initially. As sulfite treatment does not prevent bacterialgrowth (McConnell, 1991), the whiteness yielded by sulfite treatment isshort-lived. Sulfite-treated mushroom quality deteriorated markedly byday 3 (FIG. 14), and dark, sunken lesions appeared by day 6.

The hydrogen peroxide/EDTA treatment developed by McConnell (1991),improved shelf-life quality of fresh mushrooms drastically, compared tosulfite treatment. On days 3, 6, and 9, the peroxide-treated mushroomswere whiter than sulfite-treated mushrooms, and, until day 9, were freeof sunken bacterial lesions. On day 9, the lesions were smaller and, byvisual inspection, covered less of the mushroom cap surface than thoseon the sulfite-treated mushrooms. In addition, peroxide-treatedmushrooms had a dryer cap surface, vs. sulfite-treated mushrooms. In thelater stages (after day 3) of shelf-life. Initially, however,sulfite-treated mushrooms are still noticeably whiter than those treatedwith hydrogen peroxide and EDTA, both by visual inspection and byreflectance colorimetry.

In terms of performance, the ideal mushroom preservative treatment(barring a theoretical one of infinite whiteness and shelf life) wouldyield an initial whiteness equal to or greater than that achieved bysulfite treatment, and would maintain whiteness throughout shelf life atleast as effectively as treatment with hydrogen peroxide and EDTA. Theoptimal high-pH treatment (0.05M sodium bicarbonate at pH 11.0, 25° C.,30s/0.6% erythorbic acid+2.4% sodium erythorbate+1000 ppm EDTA+1000 ppmcalcium chloride, 10° C., 60s) was evaluated for overall performance vs.sulfite treatment and hydrogen peroxide/EDTA treatment. L-value(whiteness) measurements and visual observations were recorded on days0, 3, 6, and 9, and results are shown in FIGS. 14 and 15.

On day 0, the high-pH treatment yielded the highest numerical whitenessvalue, with a 6-replicate average of L=92.32, though this was notdifferent (p<0.05) from the sulfite treatment mean of L=91.96. Theperoxide-treated mushrooms were less white, at L=89.97. On day 3, thehigh-ph-treated mushrooms were whiter than the peroxide-treatedmushrooms, which were whiter than the sulfite-treated mushrooms. On days6 and 9, the high-pH and peroxide treatments were equally effective, andboth outperformed sulfite treatment by more than 10L-value units. Thesulfite-treated mushrooms were visibly slimy and had sunken lesions byday 6. By day 9, the lesions were dark brown to black and covered mostor all of the mushroom cap surfaces. The peroxide- and high-pH-treatedmushrooms were free of blotch discoloration and sunken lesions throughday 6, and showed only mild purple to light tan blotches over part ofthe cap surface on day 9. On day 6, there was some browning visible onthe underside of the cap and on the cut end of the stripe, becomingslightly darker by day 9. The rate of discoloration was not different,at p<0.05, for the high-pH and hydrogen peroxide treatments, whereassulfite-treated mushrooms discolored much more rapidly over the 9-dayshelf life.

In summary, the high-pH treatment yielded mushrooms of equal or higherquality, vs. the sulfite and hydrogen peroxide treatments, on each dayof evaluation. Initial performance matched that of sulfites, andperformance at the end of shelf life, on days 6 and 9, matched that ofthe hydrogen peroxide/EDTA wash. Between day 0 and day 6, when freshmushrooms are typically displayed for retail sale, the high-pH treatedmushrooms were of higher quality than both sulfite-treated andperoxide-washed mushrooms, based on day-3 data.

Applications in Canning and Freezing

Though consumption of canned mushrooms is declining, canning remainseconomically important to the mushroom industry. With the beneficialeffect of high-pH treatment on the quality and shelf life of freshmushrooms, it was investigated whether there was a similar benefit tohigh-pH treatment of mushrooms prior to canning or freezing.

Canning

Mushrooms are commonly washed and stored for 1-2 days before canning, toimprove yield (Beelman, 1997). The longer mushrooms are stored, thegreater the yield improvement (Beelman, 1997); however, color declines.Therefore, canners sometimes wash mushrooms in sulfites to preventbrowning. Thus, it was determined whether washing mushrooms in the highpH/neutralization wash would yield color as good as or better than thatof a sulfite treatment, while still providing the yield benefit ofwashing and holding.

Canned mushrooms were washed in reverse-osmosis water, a sulfitesolution, or the high-pH/erythorbate solutions prior to blanching,canning, and thermal processing. Mushrooms were stored at roomtemperature and cans were opened after 7 days, to evaluate color andyield. Color results are given in Table 7. High-pH mushrooms weresignificantly whiter than sulfite-treated mushrooms (by a difference ofapproximately 3 L-value points), which were significantly whiter thanthe water-washed mushrooms.

Yield was calculated as a percentage of fresh weight. Results are givenin Table 8. Sulfite treatment and high-pH treatment resulted in similaryields (65.70% and 65.53%, respectively), while water washing resultedin a slightly, but significantly, lower yield of 64.85%.

Since the high-pH wash protected the mushrooms from browning duringstorage better than sulfites, these mushrooms could perhaps have beenstored longer prior to canning to result in even greater canned productyield without sacrificing color.

Freezing

Frozen mushroom color was evaluated at 2, 4, 6, and 8 weeks afterfreezing, and coliform and total aerobic plate counts were determined.Frozen mushrooms pre-treated with the high-pH/erythorbate wash were muchwhiter than mushrooms pre-washed in water or in 1000 ppm sodiummetabisulfite, 2, 4, 6, and 8 weeks after washing and freezing. Frozenmushroom color results are given in FIG. 16.

Bacterial growth on frozen mushrooms was reduced by high-pHpre-treatment (FIG. 17). After six weeks of frozen storage, aerobicplate counts on sulfite-washed mushrooms were higher than those onwater-washed mushrooms, but on all four weeks of evaluation, platecounts were lowest for the high pH-washed mushrooms. Coliform countswere<10 CFU/g through 8 weeks of frozen storage for the high-pHtreatment. They were similar for water-washed mushrooms, but were ashigh as 375 CFU/g for sulfite-washed mushrooms (Table 9).

CONCLUSIONS

A two-stage wash treatment consisting of a 0.05M sodium bicarbonatebuffer at pH 10.5-11.0 in the first stage, followed by a neutralizationsolution containing 0.6% erythorbic acid, 2.4% solution erythorbate,1000 ppm EDTA, and 1000 ppm calcium chloride in the second stage is veryeffective at improving shelf life and quality of fresh and processedwhite mushrooms (Agaricus bisporus). This treatment equals the initialwhiteness achieved by sulfite treatment, while controlling bacterialgrowth, preventing blotch and lesion formation, and improving shelf lifeand storage quality as effectively at or better than wash treatmentsincorporating hydrogen peroxide and EDTA.

Wash solution temperatures and mushroom holding times in wash solutionsaffect the performance of the high-pH/erythorbate treatment. A retentiontime of 30 seconds in a pH 10.5-11.0 first-stage buffer at 25° C.,followed by 60 seconds in a 3% erythorbate solution at 10° C. weredetermined to be optimal processing conditions. The treatment was foundto be robust, however, and was effective over a range of temperatures,holding times, and even wash solution ingredient concentrations. The pHof the first-stage wash solution could be reduced to 9.5-10.0 withoutserious detriment to performance, particularly if the buffering capacity(sodium bicarbonate concentration) is increased. Similarly, theerythorbic acid concentration could be reduced to as low as 0.4% andsodium erythorbate concentration as low as 1.6% (retaining the 1:4erythorbic acid: sodium erythorbate ratio) in the second-stage wash.

The addition of 1000 ppm EDTA and 1000 ppm calcium chloride to thesecond-stage wash solution enhanced the performance of the treatment,with each ingredient resulting in an improvement in mushroom color. EDTAfunctions to chelate copper, a cofactor of polyphenol oxidase, thebrowning enzyme in mushrooms. It has also been shown to enhance theperformance of antimicrobials. Calcium chloride may function byincreasing solute concentration at the mushroom cap surface, making lesswater available to bacteria and increasing surface light reflectance(whiteness). In addition, it may improve vacuolar membrane integrity,making the mushroom tissue more resistant to bruising and senescence.

The high pH of the first-stage wash is designed to destroy bacteria onthe mushroom cap surface, particularly the phytopathogenic fluorescentpseudomonads, which cause blotches and lesions. Erythorbic acid andsodium erythorbate, in addition to returning mushroom pH tophysiological range, act as antioxidants, inhibiting enzymatic browning.

In addition to effectively improving the quality and shelf life of freshmushrooms, high-pH/erythorbate treatment is useful as a pretreatment toimprove the color of canned and frozen mushrooms.

TABLE 1 Neutralization Solution Formulations and pH Readings. pH with pHwith % Equal Vol. Equal Vol. Total Initial NaHCO₃ pH TSP @ SolutionSolute pH @ pH 11.0 pH 11.0 1% Sodium Erythorbate 1 8.35 10.75 11.13 2%Sodium Erythorbate 2 8.31 10.56 11.06 3% Sodium Erythorbate 3 8.31 10.5210.99 4% Sodium Erythorbate 4 8.29 10.45 10.96 1:4 E.A.:Na Erythorbate 15.18 10.42 11.09 1:3 E.A.:Na Erythorbate 1 5.01 10.13 10.85 1:1 E.A.:NaErythorbate 1 3.87 9.60 10.70 3:1 E.A.:Na Erythorbate 1 3.39 8.82 10.581:4 E.A.:Na Erythorbate 2 5.02 10.34 11.02 1:3 E.A.:Na Erythorbate 24.85 10.06 10.88 1:1 E.A.:Na Erythorbate 2 4.17 7.02 10.68 3:1 E.A.:NaErythorbate 2 3.43 5.72 9.69 1:4 E.A.:Na Erythorbate 3 4.53 6.91 10.711:3 E.A.:Na Erythorbate 3 4.46 6.83 10.49 1:1 E.A.:Na Erythorbate 3 4.205.99 9.85 3:1 E.A.:Na Erythorbate 3 3.98 5.00 8.30 1:4 E.A.:NaErythorbate 4 4.98 7.28 10.66 1:3 E.A.:Na Erythorbate 4 4.82 6.98 10.531:1 E.A.:Na Erythorbate 4 4.29 5.25 8.30 3:1 E.A.:Na Erythorbate 4 3.694.60 7.60 1% Erythorbic Acid 1 2.72 6.73 10.49 2% Erythorbic Acid 2 2.645.99 9.34 3% Erythorbic Acid 3 2.55 3.82 7.48 4% Erythorbic Acid 4 2.533.68 7.12 E.A. = Erythorbic acid. Na Erythorbate = Sodium Erythorbate.TSP = Tribasic Sodium Phosphate.

TABLE 2 Chemicals Used in the Mushroom Wash Treatments and TheirSources. Calcium-disodium EDTA (Vernene ® CA) The Dow Chemical Co., foodgrade Midland, MI Calcium chloride, dihydrate (Dow Flake ®) The DowChemical Co., Midland, MI Erythorbic acid, FCC fine granular Pfizer,Inc., New York, NY Hydrogen peroxide, 35% Fisher Scientific, Inc., FairLawn, NJ Sodium bicarbonate, anhydrous, Certified ACS Fisher Scientific,Inc., Fair Lawn, NJ Sodium carbonate, anhydrous, Certified ACS FisherScientific, Inc., Fair Lawn, NJ Sodium erythorbate, FCC granular Pfizer,Inc., New York, NY Sodium hydroxide, Certified ACS Fisher Scientific,Inc., Fair Lawn, NJ Sodium sulfate, anhydrous, Certified ACS FisherScientific, Inc., Fair Lawn, NJ

TABLE 3 Influence of calcium chloride added to the second-stage washsolution on the color of hybrid off-white mushrooms. L-value TreatmentDay 0 Day 3 Day 6 Day 9 Water Control 92.61b 91.68c 86.43b 82.83c pH11.0 no Ca 93.95a 94.58b 92.57a 89.06b ph 11.0 + Ca 94.22a 95.09a 92.88a90.69a Data are means of four replicates: within each day of evaluation,means followed by the same letter are not significantly different (P <0.05).

TABLE 4 Influence of calcium chloride added to the second-stage washsolution on the bacterial population of fresh mushrooms. CPU/mlTreatment Day 0 Day 3 Day 6 Day 9 Water Control  3.4 × 10²a 1.66 × 10²a7.86 × 10²a 3.38 × 10²a pH 11.0 no Ca 2.07 × 10⁴c 2.09 × 10⁴b 1.54 ×10⁴b 2.04 × 10⁴b pH 11.0 + Ca 2.31 × 10⁴b 2.20 × 10⁷b 1.53 × 10⁴b 1.45 ×10⁴c Within each day of evaluation, means followed by the same letterare not significantly different (P < 0.05).

TABLE 5 Effects of temperatures on wash solutions and holding times onwater uptake of mushrooms. Water- Weight Treatment Gain (%) 1. pH 11.0,10° C., 60 seconds/neutralization, 10° C. 11.30 (A) 120 seconds 2. pH11.0, 15° C., 30 seconds/neutralization, 25° C. 10.22 (B) 60 seconds 3.pH 11.0, 10° C., 30 seconds/neutralization, 10° C. 9.96 (B) 60 seconds4. R.O. Water, 10° C., 180 seconds 9.50 (BC) 5. pH 11.0, 35° C., 30seconds/neutralization, 10° C. 8.75 (C) 60 seconds 6. R.O. Water, 10°C., 90 seconds 8.25 (CD) 7. pH 11.0, 25° C., 30 seconds/neutralization,10° C. 7.65 (D) 60 seconds

TABLE 6 Influence of Wash Treatment Upon the Texture of Fresh Mushrooms.Resistance Treatment (Kg) 1. Unwashed Control 0.572 (A) 2. R.O. Water,90 s 0.570 (A) 3. 1000 ppm Sodium Metabisulfite, 90 a 0.567 (A) 4. pH11.0, 30 a/Neutralization*, 60 a 0.556 (A) 5. 1000 ppm HydrogenPeroxide + 1000 ppm EDTA, 90 a 0.546 (A) *Neutralization wash = 0.6%erythorbic acid + 2.4% sodium erythorbate + 1000 ppm EDTA + 1000 ppmcalcium chloride. Values are means of three replicates. Means followedby the same letter are not different at p < 0.005.

TABLE 7 Quality of Canned Mushrooms: High-pH treatment vs. Sulfite andR.O. Water Treatments. Treatment Whiteness (L-value) High-pH 64.01 (A)Sulfite 61.23 (B) R.O. Water 59.13 (C) Values are the means of fourreplications. Means followed by the same letter are not significantlydifferent at p < 0.05.

TABLE 8 Canning Yield for Washed Mushrooms: High-pH Treatment vs.Sulfite and R.O. Water Treatments. Treatment Canning Yield (%)* Sulfite65.70 (A) High-pH 65.53 (A) R.O. Water 64.85 (B) *Canning yield wascomputed on a fresh-weight basis. Values are means of four replicates.Means followed by the same letter are not significantly different at p <0.05.

TABLE 9 Coliform Counts on Mushrooms Washed Before Freezing. High-pHTreatment vs. Sulfite and R.O. Water Treatments. Coliform Count (CFU/g)Treatment 2 weeks 4 weeks 6 weeks 8 weeks Sulfite 120 375 30 10 R.O.Water <10 <10 10 10 High pH <10 <10 <10 <10 Values are means of threereplicate places each of 10⁻¹, 10⁻², and 10⁻³ dilutions.

APPENDIX TABLE 1 Effect of a Trisodium Phosphate (TSP) Wash on theStorage Quality of Fresh Mushrooms. Whiteness (L-value) Treatment Day 0Day 3 Day 6 1. Unwashed Control 90.39 87.32 81.33 2. R.O. Water, 120 a93.36 91.60 86.61 3. 1000 ppm Sodium Metabisulfite, 120 a 95.10 92.6389.53 4. 10% Trisodium Phosphate, 120 a 60.42 58.84 58.91

APPENDIX TABLE 2 Influence of Reduced TSP Concentration and aNeutralization Wash on the Performance of a TSP Mushroom PreservativeTreatment. Whiteness (L-value) Treatment Day 0 Day 3 Day 6  1. R.O.Water, 120 a 87.89 85.89 78.92  2. 1000 ppm Sodium Metabisulfite, 120 a93.16 90.75 82.75  3. 10% Trisodium Phosphate (TSP), 120 a 72.45 70.5067.51  4. 10% TSP, 60 s, R.O. Water, 60 a 80.22 85.32 76.67  5. 10% TSP,60 s, 4.50% E.A., 60 a 90.82 91.00 89.50  6. 10% TSP, 60 s, 2.25% NaE,60 a 89.23 87.67 84.32  7. 10% TSP, 60 s, 2.25% E.A., 60 a 90.71 90.9184.12  8. 5% TSP, 60 s, 2.25% E.A., 60 a 87.92 86.92 78.60  9. 2.5% TSP,60 s, 2.25% E.A., 60 a 89.59 87.38 77.90 10. 2.5% TSP, 60 s, 1.00% E.A.,60 a 88.35 85.06 76.47 E.A. = erythorbic acid NaE = sodium erythorbate

APPENDIX TABLE 3 Evaluation of TSP-vs. Sodium Bicarbonate-Based High-pHPreservative Treatments. Whiteness (L-value) Treatment Day 0 Day 3 Day6 1. R.O. Water, 120 a 86.63 82.23 78.08 2. 1000 ppm SodiumMetabisulfite, 120 a 94.52 91.23 83.78 3. 10% TSP, 60 s; 4.50% E.A., 60a 87.97 85.64 81.75 4. 10% TSP, 60 s; 2.25% E.A., 60 a 87.45 83.93 79.655. 5% NaHCO₂, 60 s; 2.25% E.A., 60 a 88.62 85.87 83.05 6. 0.05M NaHCO₂,60 s, 0.2% E.A. 60 a 92.66 92.90 89.10

1. A method for preserving fresh and processed mushrooms, comprising thesteps of: contacting the mushrooms with an antimicrobial buffer solutionhaving a pH of from about 9.5 to about 11.0; and rinsing the mushroomsone or more times immediately after said contacting step withpH-neutralizing buffer solutions of erythorbic acid and sodiumerythorbate, in ratios of about 1:4, having a sufficient pH to returnthe mushrooms to the mushroom physiological pH of about 6.5.
 2. Themethod of claim 1 wherein said antimicrobial solution is 0.05-0.5Msodium bicarbonate buffer solution, and the pH-neutralizing buffersolutions are about 0.04-0.6% erythorbic acid and about 1.6-2.4% sodiumerythorbate.
 3. The method of claim 2 wherein said contacting step iscarried out for about 30-60 seconds at about 10-35° C., and said rinsingstep is carried out for about 60-120 seconds at about 10-25° C.
 4. Themethod of claim 3 wherein said pH-neutralizing buffer solutions furtherinclude 1000 ppm calcium-disodium EDTA.
 5. The method of claim 3 whereinsaid pH-neutralizing buffer solutions further include 1000 ppm calciumchloride.
 6. The method of claim 3 wherein said pH-neutralizing buffersolutions further include 1000 ppm calcium-disodium EDTA and 1000 ppmcalcium chloride.
 7. The method of claims 2-6 wherein said antimicrobialsolution is a 0.05M sodium bicarbonate buffer solution having a pH ofabout 10.5-11.0, and the pH-neutralizing buffer solutions include about0.6% erythorbic acid and about 2.4% sodium erythorbate, and saidcontacting step is carried out for about 30 seconds at about 25° C. andsaid rinsing step is carried out for about 60 seconds at about 10° C. 8.The method of claim 1 wherein said antimicrobial solution is a 5-10%tribasic sodium phosphate solution.
 9. A method for preserving fresh andprocessed mushrooms, comprising the steps of: contacting the mushroomswith an antimicrobial buffer solution having a pH of at least about 9but not so high as to cause chemical damage to mushroom tissue; rinsingthe mushrooms one or more times after said contacting step with apH-neutralizing/browning inhibitor solution comprising erythorbic acid,sodium erythorbate, or combinations thereof, wherein the mushrooms arereturned to mushroom physiological pH.
 10. The method of claim 9 whereinthe antimicrobial buffer solution has a pH in the range of about 10.5 toabout 11.0.
 11. The method of claim 9 wherein the pH-neutralizingsolution comprises a browning inhibitor.
 12. The method of claim 9wherein the pH-neutralizing solution comprises erythorbic acid.
 13. Themethod of claim 9 wherein the pH-neutralizing solution comprises sodiumerythorbate.
 14. The method of claim 9 wherein the pH-neutralizingsolution is a buffer solution comprising erythorbic acid and sodiumerythorbate.
 15. The method of claim 14 wherein the pH-neutralizingsolution is a buffer solution comprising about 0.04-0.6 % erythorbicacid and about 1.6-2.4 % sodium erythorbate.
 16. The method of claim 9wherein the pH-neutralizing solution comprises EDTA.
 17. The method ofclaim 14 wherein the pH-neutralizing solution comprises EDTA.
 18. Themethod of claim 9 wherein the pH-neutralizing solution comprises calciumchloride.
 19. The method of claim 14 wherein the pH-neutralizingsolution comprises calcium chloride.
 20. The method of claim 9 whereinthe pH-neutralizing solution comprises EDTA and calcium chloride. 21.The method of claim 14 wherein the pH-neutralizing solution comprisesEDTA and calcium chloride.
 22. The method of claim 9 wherein thepH-neutralizing solution is water.
 23. The method of claim 9 wherein theantimicrobial buffer solution comprises sodium bicarbonate.
 24. Themethod of claim 10 wherein the antimicrobial buffer solution comprisessodium bicarbonate.
 25. The method of claim 11 wherein the antimicrobialbuffer solution comprises sodium bicarbonate.
 26. The method of claim 14wherein the antimicrobial buffer solution comprises sodium bicarbonate.27. The method of claim 17 wherein the antimicrobial buffer solutioncomprises sodium bicarbonate.
 28. The method of claim 19 wherein theantimicrobial buffer solution comprises sodium bicarbonate.
 29. Themethod of claim 21 wherein the antimicrobial buffer solution comprisessodium bicarbonate.
 30. The method of claim 9 wherein the antimicrobialbuffer solution comprises tribasic sodium phosphate.
 31. The method ofclaim 10 wherein the antimicrobial buffer solution comprises tribasicsodium phosphate.
 32. The method of claim 11 wherein the antimicrobialbuffer solution comprises tribasic sodium phosphate.
 33. The method ofclaim 14 wherein the antimicrobial buffer solution comprises tribasicsodium phosphate.
 34. The method of claim 17 wherein the antimicrobialbuffer solution comprises tribasic sodium phosphate.
 35. The method ofclaim 19 wherein the antimicrobial buffer solution comprises tribasicsodium phosphate.
 36. The method of claim 21 wherein the antimicrobialbuffer solution comprises tribasic sodium phosphate.
 37. The method ofclaim 22 wherein the antimicrobial buffer solution comprises tribasicsodium phosphate.
 38. The method of claim 9, wherein the pH of theantimicrobial buffer solution is about 9 to about 11.